Protein having PGE2 synthase activity and use thereof

ABSTRACT

A single protein which is the substance of the PGE2 synthesis activity in brain soluble fractions of LPS administered rats has been purified and identified. The protein has an activity of synthesizing PGE2 from PGH2, and further, has an activity of synthesizing PGE2 from arachidonic acid in combination with COX.

TECHNICAL FIELD

This invention relates to proteins having PGE2 synthase activity and usethereof.

BACKGROUND ART

Nonsteroidal anti-inflammatory drugs (NSAIDs), represented by aspirinand piroxicam, are used widely as antipyretic analgesic antiphlogisticdrugs and are considered to manifest anti-inflammatory effects, such asantipyretic, analgesic, and antiphlogistic effects, by suppressing theproduction of prostaglandins through inhibitory effects on COX(cyclooxygenase). COXs are enzymes that produce PGH2 (prostaglandin H2)from arachidonic acid, and 2 types of enzymes, referred to asconstitutive COX-1 and inducible COX-2, are known to exist. Depending onthe cell or tissue of production, PGH2 produced by COX is enzymaticallyconverted to PGE2, PGD2, PGF2α, PGI2 (prostacyclin), or TXA2(thromboxane A2) (Vane J R and Botting R M, Inflammation Research. 44:1,1995).

In particular, suppression of PGE2 production among the above-mentionedprostanoids is suggested to be important for NSAIDs to show the effectof the drug, due to the fact that PEG2 among these prostanoids isbelieved to be deeply involved with inflammatory processes, such as paingeneration, fever, and edema, and that it exists as the highestconcentration at the site of inflammation (Vane J R and Botting R M,Inflammation Research. 44:1, 1995); moreover, the drug efficacies ofanti-PGE2 antibody and NSAIDs are reported to be nearly equal in a ratinflammatory model (Portanova J P, Zhang Y, Anderson G D, Hauser S D,Masferrer J L, Seibert K, Gregory S A, Isakson P C, Journal ofExperimental Medicine. 184:883-91, 1996).

On the other hand, NSAIDs, apart from PGE2, also suppress the productionof PGD2, PGF2α, PGI2, and TXA2 by inhibiting COX, and thus, may exhibitnot only anti-inflammatory effects but also effects based on thesuppression of the production of these other prostanoids. For example,childbirth is known to be delayed by the inhibition of uterinecontraction at the time of delivery due to suppression of PGF2αproduction, and blood coagulation is known to be delayed by thesuppression of TXA2 production (AHFS Drug Information 98, p 1571 McEnvoyG K Ed., American Society of Health-System Pharmacists, 1998).

Therefore, substances that specifically inhibit the action of PGE2, suchas PGE2 synthase (PGES) inhibitors, are expected to serve as excellentanti-inflammatory drugs with lower side effects, by specificallysuppressing PGE2 production without suppressing the production of otherprostanoids.

To date, although synthases specific for PGD2, PGF2α, PGI2, and TXA2,respectively, have been identified, those for PGE2 synthase have beensuggested to exist but have not yet been identified.

Recently, Per-Johan Jakobsson et al. identified a membrane-bound humanPGE2 synthase for the first time (Proc. Natl. Acad. Sci. U.S.A.,96:7220-7225, 1999) (herein, the enzyme is referred to as “PGES-2”).

On the other hand, the existence of other enzymes with characteristicsdifferent from the PGES-2 has been also suggested. Specifically,Ogorochi et al. have estimated that PGES-2 and a protein of pI5.4 withPEGS activity existing in the cytoplasm of human brain are identical,because the proteins are purified together with glutathioneS-transferase (GST) (Ogorochi T, Ujihara M., and Narumiya S., J.Neurochem., 48:900-909, 1987).

DISCLOSURE OF THE INVENTION

The object of the present invention is to identify proteins having PGE2synthase activity, and to provide a use for the proteins. According toone embodiment, the present invention provides methods for producingPGE2 using the protein. In another embodiment, methods for screeningPGE2 synthase inhibitors using cells expressing the protein areprovided. According to a preferred embodiment, the invention providesmethods for screening PGE2 synthase inhibitors using cells co-expressingPGE2 synthase and COX.

The present inventors discovered that the ability to synthesize PGE2 isremarkably induced in the soluble fraction of LPS-administered ratbrain. Based on this finding, the present inventors vigorously conductedresearch to identify the substance responsible for the PGE2 synthesisactivity in these fractions. And as a result, the inventors succeeded inidentifying and purifying a single protein, which is the substanceresponsible for the activity.

A database search using the amino acid sequence of the obtained proteindemonstrated a surprising result, which demonstrated the protein to beidentical to a protein reported as human progesterone receptor complexcomponent protein (Johnson J L et al., Mol. Cell. Biol., 14:1956-63,1994).

Further, a cDNA encoding the obtained protein was inserted into anexpression vector to transfect HEK293 cells. A glutathione(GSH)-dependent PGE2 synthesis activity (PGES activity) was observed inthe cell lysate of the transfected cells, and the activity wassuppressed by 1-chloro-2,4-dinitrobenzene (CDNB), a GST inhibitor. Thus,the cDNA isolated by the present inventors was confirmed to encode theobject PGES (the protein was dubbed “PGES-1”). The protein, which hadbeen reported as progesterone receptor complex component protein, wasdemonstrated to be a protein belonging to PGES.

In addition, the present inventors discovered that screening for PGESinhibitors is enabled by utilizing a system that produces PGE2 from PGH2using cells made to express PGES-1. However, PGH2, a direct substrate ofPGES, is extremely chemically unstable and decomposes non-enzymatically.Thus, construction of a system for screening PGES inhibitors, which ismore stable than that wherein PGH2 is added to PGES-expressing cells, isdesired in the art. The present inventors presumed that a stablescreening system may be constructed by producing human cells thatsimultaneously express human PGES and human COX, and adding arachidonicacid, a relatively stable substrate of COX, to these cells.

Accordingly, first, the relationship of PGES-1 to COX-1 and COX-2 wereexamined. The inventors observed that PGE2 production levels increaseddrastically in the presence of arachidonic acid in cells prepared bytransfecting human PGES-1 cDNA to HEK293 cells that express human COX-1as compared cells without human PGES-1 cDNA transfection. In contrast,human COX-2-expressing HEK293 cells that were made to express PGES-1 didnot show an increase in PGE2 production. Cooperative function (coupledfunction) of COX-1 and PGES-1 was demonstrated. Furthermore, aphenomenon where both enzymes function cooperatively (coupling) was alsoconfirmed in cells expressing both COX-2 and PGES-2. Thus, the presentinventors succeeded in establishing a system that produces PGE2 fromarachidonic acid by utilizing cells made to express both COX and PGES.This system enables the efficient screening of PGES inhibitors.Compounds isolated by such screening are expected to be applicable asanti-inflammatory drugs and such.

The present invention relates to proteins having PGE2 synthase activity,as well as to methods for producing PGE2 and screening PGE2 synthaseinhibitors utilizing the PGE2 synthase activity. Specifically, thepresent invention provides:

(1) a protein having PGE2 synthase activity, comprising the amino acidsequence of SEQ ID NO: 1;

(2) a protein having PGE2 synthase activity selected from the group of:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 1 inwhich one or more amino acids are substituted, deleted, added, and/orinserted; and

(b) a protein encoded by a DNA that hybridizes under stringentconditions to a DNA consisting of the nucleotide sequence of SEQ ID NO:1;

(3) the protein of (1) or (2), which is used to synthesize PGE2;

(4) a DNA that encodes the protein of (1) or (2);

(5) a vector containing the DNA of (4);

(6) a transformant carrying the vector of (5);

(7) a method for producing the proteins of (1) or (2), comprising thesteps of cultivating the transformant of (6), and collecting theexpressed protein from said transformant or from the culture supernatantthereof;

(8) a method for producing PGE2, wherein the protein of (1) or (2) isacted on PGH2;

(9) a method for producing PGE2, wherein COX and PGE2 synthase are actedon arachidonic acid;

(10) the method of (9), wherein COX is COX-1 and PGE2 synthase is theprotein of (1) or (2);

(11) the method of (9), wherein COX is COX-2 and PGE2 synthase isPGES-2;

(12) a PGE2 synthesizing agent containing the protein of (1) or (2) asthe active ingredient;

(13) a transformant carrying a vector containing a DNA encoding COX anda vector containing a DNA encoding PGE2 synthase;

(14) the transformant of (13), wherein COX is COX-1 and PGE2 synthase isa protein of (1) or (2);

(15) the transformant of (13), wherein COX is COX-2 and PGE2 synthase isPGES-2;

(16) a method of screening for PGE2 synthase inhibitors, comprising thesteps of:

(a) contacting the transformant of (6) with a test sample and PGH2;

(b) detecting the level of PGE2 produced by said transformant; and

(c) selecting the compound that reduces the level of PGE2 produced ascompared with that produced in the absence of the test sample;

(17) a method for screening PGE2 synthase inhibitors, comprising thesteps of:

(a) contacting the transformant of any one of (13) to (15) with a testsample and arachidonic acid;

(b) detecting the level of PGE2 produced by said transformant; and

(c) selecting the compound that reduces the level of PGE2 produced ascompared with that produced in the absence of the test sample;

(18) a PGE2 synthase inhibitor that can be isolated by the screeningmethod of (16) or (17); and

(19) an anti-inflammatory drug containing the PGE2 synthase inhibitor of(18) as the active ingredient.

Herein, the term “PGE2 synthase” refers to enzymes that have theactivity to produce PGE2 using PGH2 as substrate. Accordingly, herein,“PGE2 synthase activity” refers to an activity to produce PGE2 usingPGH2 as substrate. Moreover, the term “COX” herein refers to enzymesthat have the activity to produce PGH2 using arachidonic acid assubstrate.

The present invention provides a PGES-1 protein having PGE2 synthaseactivity. The amino acid sequence of the PGES-1 protein isolated by thepresent inventors is indicated in SEQ ID NO: 1, and the nucleotidesequence of a cDNA encoding the protein is indicated in SEQ ID NO: 2.The PGES-1 protein was isolated by purifying proteins from solublefractions of LPS-administered rat brain using the PGE2 synthesizingactivity as an index. The primary structure of the PGES-1 protein isidentical to the protein reported as human progesterone receptor complexcomponent protein (Johnson J L et al., Mol. Cell. Biol., 14:1956-63,1994); however, the present inventors were the first to find that thisprotein has PGE2 synthase activity. As described later, the PGES-1 maybe utilized to produce PGE2 and to screen PGE2 synthase inhibitors dueto the PGE2 synthase activity thereof.

The present invention includes those proteins that are structurallysimilar to the PGES-1 protein (SEQ ID NO: 1), so long as they retain thePGE2 synthase activity. Such proteins may include, for example, mutantsof the PGES-1 protein, allele variants, homologs, and such.

One method well known to those skilled in the art for preparingfunctionally equivalent proteins is to introduce mutations intoproteins. For example, one skilled in the art can prepare mutants thatretain PGE2 synthase activity of human PGES-1 proteins by introducingappropriate mutations into the amino acid sequence of the protein (SEQID NO: 1), by using site-specific mutagenesis (Hashimoto-Gotoh, T. etal. (1995) Gene 152, 271-275; Zoller, M J, and Smith, M. (1983) MethodsEnzymol. 100, 468-500; Kramer, W. et al. (1984) Nucleic Acids Res. 12,9441-9456; Kramer W, and Fritz H J (1987) Methods Enzymol. 154, 350-367;Kunkel, TA (1985) Proc Natl Acad Sci USA. 82, 488-492; Kunkel (1988)Methods Enzymol. 85, 2763-2766), and such. Mutation of amino acids mayoccur in nature as well. Thus, a protein comprising the amino acidsequence of human PGES-1 protein (SEQ ID NO:1) in which one or moreamino acids are mutated is also included in the present invention, solong as it has PGE2 synthase activity. In such a mutant protein, thenumber of amino acids mutated is typically 30 residues or less,preferably 10 residues or less, more preferably 5 residues or less (forexample 3 residues or less).

It is preferable to mutate an amino acid residue into one that allowsthe properties of the amino acid side-chain to be conserved. Examples ofproperties of amino acid side chains include: hydrophobic amino acids(A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q,G, H, K, S, T), and amino acids comprising the following side chains:aliphatic side-chains (G, A, V, L, I, P); hydroxyl group-containingside-chains (S, T, Y); sulfur atom-containing side-chains (C, M);carboxylic acid- and amide-containing side-chains (D, N, E, Q);base-containing side-chain (R, K, H); and aromatic-containingside-chains (H, F, Y, W) (The letters within parenthesis indicate theone-letter codes of amino acids).

It is well known that a protein having deletion, addition, and/orsubstitution of one or more amino acid residues in the sequence of theprotein can retain the original biological activity (Mark, D. F. et al.Proc. Natl. Acad. Sci. U.S.A. 81:5662-5666 (1984); Zoller, M. J. andSmith, M. Nucleic Acids Res. 10:6487-6500 (1982); Wang, A. et al.Science 224:1431-1433; Dalbadie-McFarland, G. et al. Proc. Natl. Acad.Sci. U.S.A. 79:6409-6413 (1982)).

In the Example of the present invention, it was demonstrated that thesubstitution of tyrosine, the 9^(th) residue from N-terminus of thePGES-1 protein, with an aspartic acid reduces the PGE2 synthaseactivity. Therefore, this tyrosine residue is suggested to be importantfor maintaining the PGE2 synthase activity.

An alternative method well known to those skilled in the art forpreparing functionally equivalent proteins is, for example, the methodutilizing the hybridization technique (Sambrook, J. et al., MolecularCloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. Press, 1989).Generally, one skilled in the art can isolate DNA highly homologous tothe whole or part of a DNA sequence encoding human PGES-1 protein (SEQID NO: 2), and then isolate a protein functionally equivalent to thehuman PGES-1 protein from those isolated DNAs. The present inventionincludes proteins encoded by DNAs that hybridize under stringentconditions to a DNA encoding the human PGES-1 protein, so long as theprotein has PGE2 synthase activity. These proteins include, for example,non-human mammalian homologues (e.g. proteins encoded by genes of mice,rats, rabbits, cattle, and such).

Stringent hybridization conditions for isolating a DNA encoding aprotein functionally equivalent to human PGES-1 protein may beappropriately selected by a person skilled in the art. For example,prehybridization is performed at 68° C. for 30 min or more using“Rapid-hyb buffer” (Amersham LIFE SCIENCE). A labeled probe is addedthereto, and hybridization is conducted by warming at 68° C. for onehour or more. Then, washing in 2×SSC, 0.01% SDS at room temperature for20 min three times; in 1×SSC, 0.1% SDS at 37° C. for 20 min three times;and then in 1×SSC, 0.1% SDS at 50° C. for 20 min twice. However, severalfactors, such as temperature and salt concentration, can influence thestringency of hybridization and one skilled in the art can suitablyselect the factors to accomplish a similar stringency.

In place of hybridization, gene amplification methods using primerssynthesized based on the sequence information of the DNA (SEQ ID NO: 2)encoding the human PGES-1 proteins, for example, the polymerase chainreaction (PCR) method, can be utilized for the isolation.

A protein encoded by a DNA isolated through the above hybridizationtechniques or gene amplification techniques normally has a high homologyto the amino acid sequence of the human PGES-1 protein (SEQ ID NO: 1).The proteins of the present invention also include proteins that have ahigh homology to the amino acid sequence of the human PGES-1 protein, solong as the protein has a PGE2 synthase activity. “Highly homologous”refers to, normally an identity of at least 25% or higher, preferably40% or higher, more preferably 60% or higher, even more preferably 80%or higher (for example, 90% or higher, 95% or higher) at the amino acidlevel. The homology of a protein can be determined by following thealgorithm in “Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad.Sci. USA 80:726-730”.

Whether a prepared protein has the PGES synthase activity or not can bedetected, for example, by a method described in Example 1.

The proteins of the present invention may have variations in the aminoacid sequence, molecular weight, isoelectric point, the presence orabsence of sugar chains, form, and so on, depending on the cell or hostused to produce it or the purification method utilized. Nevertheless, solong as the obtained protein has PGE2 synthase activity, it is withinthe scope of the present invention. For example, if a protein of thepresent invention is expressed in a prokaryotic cell, such as E. coli,the protein includes a methionine residue at the N-terminus in additionto the natural amino acid sequence of the protein. Such proteins arealso included in the proteins of the present invention.

The proteins of the present invention can be prepared as recombinantproteins or naturally occurring proteins, using methods commonly knownin the art. Alternatively, a protein of the present invention can besynthesized artificially. When the protein is a recombinant protein, itmay be produced by inserting a DNA (for example, a DNA having thenucleotide sequence of SEQ ID NO: 2) encoding a protein of the presentinvention into an appropriate expression vector, collecting thetransformant obtained by introducing the vector into an appropriate hostcell, obtaining an extract, and then purifying and preparing the proteinusing chromatography, such as ion exchange, reverse phase, or gelfiltration; or affinity chromatography using a column immobilized withantibodies against the protein of the invention; or by combining thesecolumns. Alternatively, when a protein of the invention is expressed inhost cells (e.g., animal cells or E. coli) as a fusion protein withglutathione S transferase protein, or a recombinant protein withmultiple histidine residues, the expressed recombinant protein can bepurified using a glutathione column or nickel column. After the fusionprotein is purified, if necessary, regions of the fusion protein (apartfrom the desired protein) can be digested and removed with thrombin,factor Xa, etc.

The native protein of the invention can be isolated by methods wellknown in the art, for example, by purifying an extract of tissues orcells that express a protein of the invention with an affinity columnbound using antibodies that bind to a protein of the present invention.The antibodies may be polyclonal or monoclonal antibodies.

The proteins of the invention can be used as PGE2 synthesizing agents.The term “PGE2 synthesizing agents” herein refers to reagents forindustrial synthesis of PGE2 or for synthesis of PGE2 for research, andalso includes pharmaceutical agents for administration to living bodies.

In addition to being utilized in the above-described in vivo or in vitroproduction of a protein of the present invention, a DNA encoding aprotein of the present invention may also be applied, for example, inthe therapy of diseases caused by an aberration in the PGES synthaseactivity of a protein of the present invention. Any type of DNA, such ascDNA synthesized from mRNA, genomic DNA, or chemical synthetic DNA, canbe used, so long as the DNA encodes a protein of the present invention.Further, so long as they can encode a protein of the present invention,DNAs comprising arbitrary sequences based on the degeneracy of thegenetic code are also included.

The DNA of the present invention can be prepared using methods known inthe art. For example, a cDNA library can be constructed from cellsexpressing a protein of the present invention and hybridization can beconducted using a part of the DNA sequence of the present invention (forexample, SEQ ID NO: 2) as a probe. The cDNA library may be prepared, forexample, according to the method described by Sambrook J. et al.(Molecular Cloning, Cold Spring Harbor Laboratory Press (1989)), orinstead, commercially available DNA libraries may be used.Alternatively, a DNA of the present invention can be obtained bypreparing RNA from cells expressing a protein of the present invention,synthesizing cDNA therefrom using a reverse transcriptase, synthesizingoligo-DNA based on a DNA sequence of the present invention (for example,SEQ ID NO: 2), and amplifying the cDNA encoding a protein of the presentinvention by PCR using the oligo-DNA as primers.

From the nucleotide sequence of the obtained cDNA, one can determine anopen reading frame, and thereby, obtain the amino acid sequence of aprotein of the invention. The cDNA obtained may also be used as a probefor screening a genomic DNA library to isolate genomic DNA.

More specifically, mRNA may first be isolated from a cell, tissue, ororgan in which a protein of the invention is expressed. Known methodscan be used to isolate mRNA; for instance, total RNA may be prepared bythe guanidine ultracentrifugation (Chirgwin, J. M. et al., Biochemistry18:5294-5299 (1979)) or the AGPC method (Chomczynski, P. and Sacchi, N.,Anal. Biochem. 162:156-159 (1987)), and mRNA may be purified from totalRNA using mRNA Purification Kit (Pharmacia) and such. Alternatively,mRNA may be directly prepared by QuickPrep mRNA Purification Kit(Pharmacia).

The obtained mRNA is used to synthesize cDNA using reversetranscriptase. cDNA may be synthesized using a kit, such as AMV ReverseTranscriptase First-strand cDNA Synthesis Kit (Seikagaku Kogyo).Alternatively, cDNA may be synthesized and amplified following the5′-RACE method (Frohman, M. A. et al., Proc. Natl. Acad. Sci. U.S.A.85:8998-9002 (1988); Belyavsky, A. et al., Nucleic Acids Res.17:2919-2932 (1989)) that uses primers and such described herein; using5′-Ampli FINDER RACE Kit (Clontech); and by polymerase chain reaction(PCR).

A desired DNA fragment is prepared from the obtained PCR products andlinked to a vector DNA. The recombinant vector is used to transform E.coli and such, and the desired recombinant vector is prepared from aselected colony. The nucleotide sequence of the desired DNA can beverified by conventional methods, such as dideoxynucleotide chaintermination.

A DNA of the invention may be designed to have a sequence that isexpressed more efficiently by taking into account the frequency of codonusage in the host used for expression (Grantham, R. et al., NucleicAcids Res. 9:r43-74 (1981)). The DNA of the present invention may bealtered by a commercially available kit or a conventional method. Forinstance, the DNA may be altered by digestion with restriction enzymes,insertion of a synthetic oligonucleotide or an appropriate DNA fragment,addition of a linker, or insertion of the initiation codon (ATG) and/orthe stop codon (TAA, TGA, or TAG), etc.

The vectors of the present invention are useful in maintaining the DNAof the present invention within the host cell, or expressing a proteinof the present invention. When E. coli is used as the host cell, thereis no limitation other than that the vector should have an “ori” toamplify and mass-produce the vector in E. coli (e.g., JM109, DH5α,HB101, or XL1Blue), and such; and a marker gene for selecting thetransformed E. coli (e.g., a drug-resistance gene selected by a drug(e.g., ampicillin, tetracycline, kanamycin, or chloramphenicol)). Forexample, M13-series vectors, pUC-series vectors, pBR322, pBluescript,pCR-Script, and such can be used. Besides the vectors, pGEM-T, pDIRECT,pT7, and so on can also be used for subcloning and excision of the cDNAas well. When a vector is used to produce a protein of the presentinvention, an expression vector is especially useful. When theexpression vector is expressed, for example, in E. coli, it should havethe above characteristics in order to be amplified in E. coli.Additionally, when E. coli, such as JM109, DH5α, HB101, or XL1-Blue, areused as the host cell, the vector should have a promoter, e.g., the lacZpromoter (Ward et. al. (1989) Nature 341:544-546; (1992) FASEB J.6:2422-2427), the araB promoter (Better et al., (1988) Science240:1041-1043), or the T7 promoter, that can efficiently promote theexpression of the desired gene in E. coli. Other examples of the vectorsare pGEX-5x-1 (Pharmacia), “QIAexpress system” (QIAGEN), pEGFP, and pET(for this vector, BL21, a strain expressing T7 RNA polymerase, ispreferably used as the host).

Further, the vector may comprise a signal sequence that inducessecretion of the polypeptide. For producing the protein in the periplasmof E. coli, the pelB signal sequence (Lei, S. P. et al., J. Bacteriol.169:4379 (1987)) may be used as the signal sequence for proteinsecretion. For example, the calcium chloride method or electroporationmay be used to introduce the vector into host cells.

Examples of vectors used to produce the proteins of the presentinvention include, for example, expression vectors other than E. coli,such as expression vectors derived from mammals (e.g., pcDNA3(Invitrogen), pEGF-BOS (Nucleic Acids Res. (1990) 18(17):5322), pEF,pCDM8), insect cells (e.g., “Bac-to-BAC baculovirus expression system”(GIBCO-BRL), pBacPAK8), plants (e.g. pMH1, pMH2), animal viruses (e.g.,pHSV, pMV, pAdexLcw), retroviruses (e.g., pZIPneo) yeasts (e.g., “PichiaExpression Kit” (Invitrogen), pNV11, SP-Q01) and Bacillus subtilis(e.g., pPL608, pKTH50).

In order to express proteins in animal cells, such as CHO, COS, andNIH3T3 cells, the vector should include a promoter necessary forexpression in such cells (e.g., the SV40 promoter (Mulligan et al.,(1979) Nature 277:108), the MMLV-LTR promoter, the EF1α promoter(Mizushima et al., (1990) Nucleic Acids Res. 18:5322), the CMV promoter,etc.). It is more preferable for the vector to additionally have amarker gene that enables selection of the transformants (for example, adrug resistance gene selected by a drug (e.g., neomycin, G418, etc.)).Examples of vectors with such characteristics include pMAM, pDR2,pBK-RSV, pBK-CMV, pOPRSV, pOP13, and so on.

Furthermore, in order to stably express the gene and to amplify the copynumber in cells, the method using CHO cells deficient in nucleic acidsynthetic pathways as the host, incorporating-into the CHO cells avector (such as pCHOI) having a DHFR gene that compensates for thedeficiency, and amplifying the vector with methotrexate (MTX) can beused. Furthermore, for transiently expressing a gene, the method thattransforms COS cells that have the gene for SV40 T antigen on thechromosome with a vector (such as pcD) having the SV40 replicationorigin can be mentioned. The replication origin may be that of apolyomavirus, adenovirus, bovine papilloma virus (BPV), and the like.Further, to amplify the gene copy number in the host cells, selectionmarkers, such as aminoglycoside transferase (APH) gene, thymidine kinase(TK) gene, E. coli xanthine-guanine phosphoribosyl transferase (Ecogpt)gene, and dihydrofolate reductase (dhfr) gene may be comprised in theexpression vector.

A DNA of the present invention can be expressed in animals by, forexample, inserting a DNA of the invention into an appropriate vector andintroducing the vector into a living body by the retrovirus method, theliposome method, the cationic liposome method, the adenovirus method,and so on. Thus, it is possible to perform gene therapy of diseasescaused by a mutation in a gene of the present invention. The vectorsused in these methods include, but are not limited to, adenovirusvectors (e.g. pAdexlcw), retrovirus vectors (e.g. pZIPneo), and so on.General techniques for gene manipulation, such as insertion of the DNAof the invention into a vector, can be performed according toconventional methods (Molecular Cloning, 5.61-5.63). Administration tothe living body may be performed according to ex vivo methods or in vivomethods.

The host cell into which the vector of the invention is introduced isnot particularly limited. For example, E. coli, various animal cells,and such, can be used. The host cell of the present invention can beused, for example, as a production system to produce and express aprotein of the present invention. Protein production systems include invitro and in vivo systems. Such production systems using eukaryoticcells or prokaryotic cells can be given as in vitro production systems.

As eukaryotic host cells, for example, animal cells, plant cells, andfungi cells can be used. Mammalian cells, for example, CHO (J. Exp. Med.(1995) 108:945), COS, 3T3, myeloma, BHK (baby hamster kidney), HeLa,Vero, amphibian cells (e.g. platanna oocytes (Valle et al., (1981)Nature 291:358-340), and insect cells (e.g. Sf9, Sf21, Tn5) are known asanimal cells. Among CHO cells, those deficient in the DHFR gene,dhfr-CHO (Proc. Natl. Acad. Sci. USA (1980) 77:4216-4220) and CHO K-1(Proc. Natl. Acad. Sci. USA (1968) 60:1275), are particularlypreferable. Among animal cells, CHO cells are particularly preferablefor mass expression. A vector can be introduced into a host cell by, forexample, the calcium phosphate method, the DEAE-dextran method, methodsusing cationic liposome DOTAP (Boehringer-Mannheim), electroporation,lipofection, etc.

As plant cells, for example, plant cells originating from Nicotianatabacum are known as protein producing systems and may be used as calluscultures. As fungal cells, yeast cells such as Saccharomyces, includingSaccharomyces cerevisiae, or filamentous fungi such as Aspergillus,including Aspergillus niger, are known.

Useful prokaryotic cells include bacterial cells. Bacterial cells, suchas E. coli, for example, JM109, DH5α, HB101, and such, as well asBacillus subtilis are known.

These cells are transformed by a desired DNA, and the resultingtransformants are cultured in vitro to obtain the protein. Transformantscan be cultured using known methods. For example, culture medium, suchas DMEM, MEM, RPMI1640, or IMDM, may be used with or without serumsupplements, such as fetal calf serum (FCS) as culture medium for animalcells. The pH of the culture medium is preferably between about 6 and 8.Such cells are typically cultured at about 30 to 40° C. for about 15 to200 hr, and the culture medium may be replaced, aerated, or stirred ifnecessary.

Animal and plant hosts may be used for in vivo production. For example,a desired DNA can be introduced into an animal or plant host. Encodedproteins are produced in vivo, and then recovered. These animal andplant hosts are included in the “host” of the present invention.

Animals to be used for the production system described above includemammals and insects. Mammals, such as goats, pigs, sheep, mice, andcattle, may be used (Vicki Glaser, SPECTRUM Biotechnology Applications(1993)). Alternatively, the mammals may be transgenic animals.

For instance, a desired DNA may be prepared as a fusion gene with a genesuch as goat β casein gene that encodes a protein specifically producedinto milk. DNA fragments comprising the fusion gene are injected intogoat embryos, which are then introduced back to female goats. Desiredproteins are then recovered from milk produced by the transgenic goats(i.e., those born from the goats that had received the modified embryos)or by their offspring. To increase the amount of milk containing theproteins produced by transgenic goats, appropriate hormones may beadministered to the transgenic goats (Ebert, K. M. et al., (1994)Bio/Technology 12:699-702).

Alternatively, insects, such as silkworm, may be used. Baculovirusesinto which a DNA encoding a desired protein has been inserted can beused to infect silkworms, and the desired protein can be recovered fromthe body fluid (Susumu, M. et al., (1985) Nature 315:592-594).

As plants, for example, tobacco can be used. When using tobacco, a DNAencoding a desired protein may be inserted into a plant expressionvector, such as pMON 530, which is introduced into bacteria, such asAgrobacterium tumefaciens. Then, the bacteria can be used to infecttobacco, such as Nicotiana tabacum, and the desired polypeptide can berecovered from the leaves (Julian, K.-C. Ma et al., (1994) Eur. J.Immunol. 24:131-138).

A protein of the present invention obtained as above may be isolatedfrom inside or outside of host (medium, etc.), and purified as asubstantially pure homogeneous protein. The method for protein isolationand purification is not limited to any specific method; in fact, anystandard method may be used. For instance, column chromatography,filters, ultrafiltration, salting out, solvent precipitation, solventextraction, distillation, immunoprecipitation, SDS-polyacrylamide gelelectrophoresis, isoelectric point electrophoresis, dialysis, andrecrystallization may be appropriately selected and combined to isolateand purify the protein.

For chromatography, for example, affinity chromatography, ion-exchangechromatography, hydrophobic chromatography, gel filtrationchromatography, reverse phase chromatography, adsorption chromatography,and such may be used (Strategies for Protein Purification andCharacterization: A Laboratory Course Manual. Ed. Daniel R. Marshak etal., Cold Spring Harbor Laboratory Press (1996)). These chromatographiesmay be performed by liquid chromatographies such as HPLC and FPLC. Thus,the present invention encompasses highly purified proteins produced bythe above methods.

A protein may be optionally modified or partially deleted by treating itwith an appropriate protein-modifying enzyme before or afterpurification. For example, trypsin, chymotrypsin, lysylendopeptidase,protein kinase, glucosidase, and such are used as protein-modifyingenzymes.

Further, the present invention provides methods for producing PGE2utilizing a protein of this invention having the PGE2 synthase activity.According to an embodiment, the method is characterized by the treatmentof PGH2 with a protein having the PGE2 synthase activity of theinvention.

A protein having the PGE2 synthase activity used to produce PGE2 may bea naturally derived protein, a recombinant protein, or an artificiallysynthesized protein. Moreover, it may be a purified protein or a proteinin an intracellularly expressed form. Furthermore, the protein may beimmobilized in a reaction system. Since a PGE2 synthase of thisinvention demonstrates a glutathione (GSH) dependent activity, additionof glutathione to the reaction system is preferred. The PGH2 used forthis reaction may be, for example, a commercial product (manufactured byCayman).

Further, a preferred embodiment of the method for producing PGE2utilizing the protein of this invention having a PGE2 synthase activityis a method characterized by reacting COX and PGE2 synthase witharachidonic acid. The method takes into consideration the fact thatPGH2, which is the direct substrate of PGE2 synthase, is chemicallyextremely unstable and decomposes non-enzymatically, and thus, producesPGE2 from arachidonic acid, which is a relatively stable precursor ofPGH2. Specifically, production of PGE2 from arachidonic acid is achievedby a cooperative action using COX, that produces PGH2 from arachidonicacid, and PGE2 synthase, that produces PGE2 from PGH2.

A PGE2 synthase of this invention and COX used to produce PGE2 may benaturally derived proteins, recombinant proteins, or artificiallysynthesized proteins. Furthermore, they may be purified proteins orproteins in intracellularly expressed form. Furthermore, the proteinsmay be immobilized in a reaction system. Since a PGE2 synthase of thisinvention demonstrates a glutathione (GSH) dependent activity, additionof glutathione to the reaction system is preferred.

PGE2 was demonstrated in the Example to be produced from arachidonicacid by the cooperation of PGES-1 with COX-1, or PGES-2 with COX-2.Therefore, preferred enzymes for use in PGE2 production include thecombination of PGES-1 and COX-1, and the combination of PGES-2 andCOX-2.

The produced PGE2 may be purified by normal means of purification, forexample, distillation under normal pressure or reduced pressure;high-speed liquid chromatography using silica gel or magnesium silicate;thin layer chromatography; column chromatography, washing;recrystallization; and such.

Furthermore, the present invention provides methods of screening forPGE2 synthase inhibitors. According to an embodiment, the screeningmethod of this invention comprises the steps of: (a) contacting atransformant which carries a vector containing a PGES-encoding DNA witha test sample and PGH2; (b) detecting the production level of PGE2 ofthe cell; and (c) selecting the compound that reduces the productionlevel of PGE2 with that observed in the absence of the contact with thetest sample.

The transformant used for the screening can be prepared, for example, byinserting a DNA encoding a PGE2 synthase into a vector, such aspcDNA3.1, and then by introducing this into cells, such as HEK293 cells.

Test samples to be contacted with the transformant include, for example,cell extracts, cell culture supernatants, microorganism fermentationproducts, marine organism extracts, plant extracts, purified or crudeproteins, peptides, non-peptide compounds, syntheticlow-molecular-weight compounds, and natural compounds; but are notlimited to these examples. The test sample may include antibodiesbinding to a PGE2 synthase, or antisense oligonucleotides suppressingthe expression of the enzyme.

According to a preferred embodiment of the screening method of thisinvention, the method comprises the steps of: (a) contacting thetransformant which carries a vector containing a COX-encoding DNA and avector containing a PGES-encoding DNA, with a test sample andarachidonic acid; (b) detecting the production level of PGE2 of thetransformant; and (c) selecting the compound that reduces the productionlevel of PGE2 compared with that observed in the absence of the contactwith the test sample.

The transformant used for the screening can be prepared, for example, byinserting a DNA encoding a PGE2 synthase and a DNA encoding COX,respectively, into vectors, such as pcDNA3.1, and then byco-transfecting them into cells, such as HEK293 cells. The genes to becotransfected preferably are exemplified by the combination of PGES-1and COX-1, and the combination of PGES-2 and COX-2. Arachidonic acid tobe contacted with the transformant may be, for example, a commercialproduct (manufactured by Sigma).

Similar to the screening method mentioned above, the test samplesinclude, for example, cell extracts, cell culture supernatants,microorganism fermentation products, marine organism extracts, plantextracts, purified or crude proteins, peptides, non-peptide compounds,synthetic low-molecular-weight compounds, and natural compounds; but arenot limited to these examples. Further, the test sample may beantibodies binding to a PGE2 synthase, or antisense oligonucleotidessuppressing the expression of the enzyme.

The PGE2 production level during the screening of the present inventioncan be measured, for example, by Enzyme Immunoassay (a kit by Cayman) Asa result, if the PGE2 production level is reduced by the contact of atest sample with the transformant, compared with that observed in theabsence of the contact, the test sample is suggested to be a compoundthat inhibits the PGE2 synthase activity.

A PGE2 synthase inhibitor, which may be isolated by a screening methodof this invention, may be utilized as an anti-inflammatory drug. NSAIDs(nonsteroidal anti-inflammatory drugs), represented by aspirin andpiroxicam, which have been used widely as antipyretic analgesicantiphlogistic drugs, suppress the production of PGD2, PGF2α, PGI2, andTXA2, in addition to PGE2 by inhibiting COX. Therefore, NSAIDs not onlypossess anti-inflammatory effects but also are likely to express effectsbased on the suppression of the production of these prostanoids (otherthan the PGE2). On the other hand, since the compounds that can beisolated by the screening of this invention are expected to specificallyinhibit a PGE2 synthase, development of medicaments with fewer sideeffects is enabled.

Furthermore, cells that co-express COX and PGE2 synthase havesignificantly faster proliferation rates as compared to the controlcells, taking on a form similar to a transformant lacking contactinhibition ability, and thus, can be considered as a model system forhuman clinical cancer suggested to be related to COX2. Therefore,compounds that can be isolated by the screening of this invention may beutilized as anti-cancer drugs.

A compound isolated by the screening of this invention can be as apharmaceutical agent for humans and other mammals, such as mice, rats,guinea-pigs, rabbits, chicken, cats, dogs, sheep, pigs, cattle, monkeys,baboons, and chimpanzees. Specifically, the isolated compound can eitheritself be directly administered to subjects or it can be formulated intoa pharmaceutical composition using known pharmaceutical preparationmethods for administration. For example, according to the need, thedrugs can be taken orally, as sugar coated tablets, capsules, elixirs,and microcapsules; or non-orally, in the form of injections of sterilesolutions or suspensions with water or any other pharmaceuticallyacceptable liquid. For example, the compounds can be formulated bymixing appropriately with pharmacologically acceptable carriers ormedium, such as, sterilized water, physiological saline, plant-oil,emulsifiers, suspending agents, surfactants, stabilizers, flavoringagents, excipients, vehicles, preservatives, and binders, in a unit doseform required for generally accepted drug implementation. The amount ofactive ingredient in these preparations makes a suitable dosage withinthe indicated range acquirable.

Examples of additives that can be mixed for tablets and capsules are,binders such as gelatin, corn starch, tragacanth gum, and arabic gum;excipients such as crystalline cellulose; swelling agents such as cornstarch, gelatin, and alginic acid; lubricants such as magnesiumstearate; sweeteners such as sucrose, lactose, or saccharin; flavoringagents such as peppermint, Gaultheria adenothrix oil, and cherry. Whenthe unit dosage form is a capsule, a liquid carrier, such as oil, canalso be included in the above ingredients. Sterile composites forinjections can be formulated following normal drug implementations usingvehicles such as distilled water used for injections.

Physiological saline, glucose, and other isotonic liquids includingadjuvants, such as D-sorbitol, D-mannnose, D-mannitol, and sodiumchloride, can be used as aqueous solutions for injections. These can beused in conjunction with suitable solubilizers, such as alcohol,specifically ethanol, polyalcohols such as propylene glycol andpolyethylene glycol, non-ionic surfactants, such as Polysorbate 80 (TM)and HCO-50.

Sesame oil or Soy-bean oil can be used as a oleaginous liquid and may beused in conjunction with benzyl benzoate or benzyl alcohol as asolubilizer or may be formulated with a buffer such as phosphate bufferand sodium acetate buffer, a pain-killer such as procaine hydrochloride,a stabilizer such as benzyl alcohol, phenol, or an anti-oxidant. Theprepared injection is generally filled into a suitable ampule.

Methods well known to one skilled in the art may be used to administer apharmaceutical compound to patients, for example as intraarterial,intravenous, subcutaneous injections and also as intranasal,transbronchial, intramuscular, percutaneous, or oral administrations.The dosage varies according to the body-weight and age of a patient, andthe administration method; however, one skilled in the art can suitablyselect the dosage. If said compound can be encoded by a DNA, the DNA canbe inserted into a vector for gene therapy to perform the therapy. Thedosage and method of administration vary according to the body-weight,age, and symptoms of a patient, but one skilled in the art can selectthem suitably.

Although varying according to the symptoms, the dose of a PGE2 synthaseinhibitor that can be isolated by the screening methods of thisinvention is generally in the range of about 0.1 mg to 1 g, preferablyabout 1.0 to 100 mg, and more preferably about 1.0 to 20 mg per day foradults (body weight: 60 kg) in the case of an oral administration.Although varying according to the subject, target organ, symptoms, andmethod of administration, a single dose of a compound for parenteraladministration is advantageous, for example, when administeredintravenously to normal adults (60 kg body weight) in the form ofinjection, in the range of about 0.01 to 300 mg, preferably about 0.1 to100 mg, and more preferably about 0.1 to 10 mg per day. Doses convertedto 60 kg body weight or per body surface area can be administered toother animals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a graph demonstrating the PGES activity in ammoniumsulfate precipitated fraction of the rat brain soluble fraction.

FIG. 2 depicts graphs demonstrating the elution profiles of the PGESactivity by DEAE-Sephacel ion exchange column chromatography.

FIG. 3 depicts graphs demonstrating the elution profiles of the PGESactivity by Superdex 200 gel filtration column chromatography (top), andphotographs demonstrating the results of SDS-PAGE analyses (bottom).

FIG. 4 depicts a graph demonstrating the effect of CDNB on the PGESactivity in each fraction obtained by Superdex 200 gel filtration columnchromatography.

FIG. 5 depicts the nucleotide sequence of p23 (PGES-1) cDNA and theamino acid sequence thereof.

FIG. 6 depicts graphs demonstrating the in vitro enzyme profile of therecombinant PGES-1 and PGES-2 proteins expressed in HEK293 cells. PGES-1(left panel) was demonstrated to be GSH dependent and CDNB sensitive;whereas PGES-2 (right panel) was demonstrated to be GSH dependent andCDNB insensitive.

FIG. 7 depicts a graph demonstrating the PGES activity of a mutantprotein (PGES-1 Y9N), wherein the ninth tyrosine from the N-terminus(Tyr⁹) is mutated to asparagine. Tyr⁹ was demonstrated to be essentialfor the PGES-1 activity.

FIG. 8 depicts graphs (left and middle panels) and photographs (righttwo panels) demonstrating the effect on PGE₂ production of theintroduced PGES-1 antisense expression vector into 3Y1 cells.

FIG. 9 depicts graphs demonstrating the conversion of added arachidonicacid into PGE₂ by PGES-1 or PGES-2. PGES-1 is demonstrated to functionby selective coupling with COX-1, and PGES-2 is demonstrated to exhibitan enhanced function by the coupling with COX-2 than with COX-1.

FIG. 10 depicts a graph demonstrating the conversion of endogenousarachidonic acid into PGE₂ by PGES-2 in HEK293 transfect cellsstimulated with IL-1β.

FIG. 11 depicts photographs demonstrating the intracellular distributionof COX-2 and PGES-2. The top panel shows COX-2 distribution, the centerpanel shows PGES-2 distribution, and the lower panel is an overlay ofboth of these distributions.

FIG. 12 depicts graphs demonstrating the effect of the coexpression ofCOX-2 and PGES-2 to enhance PGE₂ production and cell proliferation.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in more detail below by way ofExamples, but should not construed as being limited to these Examples.

Example 1 Identification of the PGES-1 Protein

PGES activity was measured as follows: Cells suspended in 20 mmol/LTris-hydrochloride buffer (pH 7.4) were homogenized using ultrasonichomogenizer (Branson Sonifier Model 200) and centrifuged at 800×g for 5minutes at 4° C., and the supernatant was used as the cell lysate in thefollowing. 0.5 μg of PGH2 was added to the lysate, which was suspendedwith 0.1 mol/L Tris-hydrochloride buffer (pH 8.0) containing 1 mmol/LGSH, with a protein content of 10 μg, was incubated for 30 seconds at24° C., and then 8 mmol/L FeCl₂ was added to terminate the reaction. Theamount of produced PGE2 was measured by Enzyme Immunoassay Kit (Cayman),and PGES activity was expressed as the amount of PGE2 produced per 30seconds, per 10 μg of protein (ng/30 sec/10 μg protein).

150 μg/kg body LPS (Salmonella minnesota: Re595) was administeredintravenously from rat tail. Rat brain was removed 2 days after theinjection and was homogenized using Potter's homogenizer at 4° C. Thehomogenate was centrifuged at 10,000×g at 4° C. for 20 minutes, and thesupernatant was collected. The supernatant was centrifuged again at100,000×g at 4° C. for 60 minutes to recover the supernatant. 60 to 80%saturated ammonium sulfate precipitation fractions having PGES activity(FIG. 1) were collected from the supernatant, and fractions having thePGES activity eluted at an NaCl concentration of around 0.5 mol/L toaround 0.8 mol/L by DEAE-Sephacel ion exchange column chromatographywere collected (FIG. 2).

Furthermore, these fractions were subjected to Superdex-200 column gelfiltration FPLC (elution buffer: 20 mmol/L Tris-hydrochloride buffer (pH7.4), 150 mmol/L NaCl), to yield 3 peaks demonstrating PGES activity: A(molecular weight of approximately 300 kDa); B (molecular weight ofapproximately 100 kDa); and C (molecular weight of approximately 50kDa). No significant differences in the peaks A and B were observed inthe PGES activity between LPS-administered rats and normal rats;whereas, about 6 times higher PGES activity of peak C was observed inLPS-administered rats compared to normal rats (FIG. 3, top panel).

A protein of a molecular weight of about 27 kDa, corresponding with thePGES activity (PGES-1), was obtained by SDS electrophoretic analysis ofpeak C (FIG. 3, bottom). The production of this protein was predicted toelevate in response to inflammatory stimulus due to the fact that largeamount of the protein was detected in LPS-administered rats than innormal rats. Furthermore, in contrast to peak A and B, peak C wasstrongly inhibited by CDNB (FIG. 4). Therefore, the protein wassuggested to be a GST-like isozyme.

Peptide mapping of PGES-1 protein was carried out using V8 protease, and3 fragments among the peptide fragments were analyzed by N-terminalamino acid sequence analyzer to determine the partial amino acidsequence thereof. According to GenBank database search with one of theseamino acid sequences, the sequence completely matched to a portion of aknown sequence reported as the human progesterone receptor complexcomponent protein p23 (Johnson, J L et al., Mol. Cell. Biol.,14:1956-63, 1994). Therefore, referring to the reported cDNA sequence,the cDNA of full-length human progesterone receptor complex componentprotein p23 was isolated by RT-PCR method using HeLa cell mRNA as thematerial (FIG. 5). PGES activity dependent to GSH was observed in thecell lysate by inserting the cDNA into expression vector pcDNA3.1, andtransfecting HEK293 cells using lipofectamine, which activity wassuppressed in the coexistence of CDNB (FIG. 6, left panel). Thus, thecDNA was confirmed to encode the desired PGES. In addition, the mutantprotein, wherein the 9th tyrosine residue from the N-terminus wassubstituted by point mutation to asparagine, did not demonstrate thePGES activity, which elucidated the tyrosine residue to be essential forthe expression of the activity (FIG. 7). On the other hand, whenpcDNA3.1-hygro, which has the entire PGES-1-encoding region cloned inreverse, was transfected to human fibroblast 3Y1, PGES-1 synthesis wasinhibited and PGE2 production and PGES activity was decreaseddemonstrating the inhibition of PGE2 production by antisense RNA (FIG.8).

Although a GSH-dependent PGES activity was confirmed in HEK293 cellstransfected with PGE2 synthase (PGES-2), an enzyme reported by Per-JohanJakobsson et al., the activity was not suppressed by the coexistence ofCDNB (FIG. 6, right panel) which demonstrates the difference betweenPGES-1 and PGES-2.

The PGES-1 protein is an enzyme that converts PGH2 produced by COX,particularly by COX-1, into PGE2, and is thought to have an importantrole in inflammatory reactions where PGE2 plays a major role.

Example 2 Preparation of Cells Coexpressing COX and PGES

Human kidney-derived HEK293 cells (human, embryo, kidney, transformedwith adenovirus 5 DNA) were purchased from ATCC (ATCC Number: CRL-1573).

Expression vectors wherein human COX-1 and COX-2 cDNAs were insertedinto pcDNA3.1, respectively, were transfected into HEK293 cells usinglipofectamine to produce HEK293 cells that express either COX-1 orCOX-2. HEK293 cells expressing PGES-1 or PGES-2 alone, or coexpressingCOX-1 and PGES-1, COX-1 and PGES-2, COX-2 and PGES-1, or COX-2 andPGES-2 were constructed by similarly transfecting expression vectorswherein either human PGES-1 cDNA or human PGES-2 cDNA was inserted intopcDNA3.1 into constructed COX-1 or COX-2 expressing cells and intoHEK293 cells of the parent strain, and these cells were used in thefollowing experiment. Evaluation of cell proliferation was performed byseeding the cell on RPMI1640 media in a 6-well plate (Iwaki) at1.5×10⁵/3 mL/well, incubating in carbon dioxide gas incubator at 5% CO₂for 4 days, and then, calculating the number of living cells by thetrypan blue staining method using a cytometric plate.

The PGE2 production level of these cells under a condition adding 2μmol/L of arachidonic acid to the culture medium was examined. The PGE2production level of cells coexpressing COX-1 and PGES-1 largelyincreased compared to cells expressing COX-1 alone, whereas no increasein PGE2 production was observed in cells coexpressing human COX-2 andPGES-1 (FIG. 9, left panel) This suggests the coupling (cooperativefunctioning) of PGES-1 and COX-1. On the other hand, large amounts ofPGE2 was produced by the expression of PGES-2 together with COX-2 (FIG.9, right panel), suggesting the coupling (cooperative functioning) ofPGES-2 and COX-2. A more remarkable increase in PGE2 production bycoupling of PGES-2 and COX-2 was demonstrated by stimulating cells with1 ng/mL human Interleukin-1β (IL-1β; Genzyme) under conditions withoutthe addition of arachidonic acid to the culture medium (FIG. 10).

Furthermore, a close relation between COX-2 and PGES-2 was alsodemonstrated by a similar distribution of the enzymes detected by anindirect immunostaining using a combination of mouse monoclonal antibodyagainst FLAG with FITC-labeled anti-mouse IgG antibody, or goat antiseraagainst COX-2 (Santa Cruz, N-20 goat polyclonal Ab) with Cy3-labeledanti-goat IgG antibody on cells, which cells were prepared to coexpressCOX-2 and a fusion protein, consisting of PGES-2 and FLAG, by similarlytransfecting an expression vector containing PGES-2 cDNA and FLAG cDNAto cells that express COX-2 alone (FIG. 11).

Not only PGES but also COX is greatly involved in the pathway to producePGE2 via the arachidonic acid metabolic pathway. Therefore, whenscreening for PGES inhibitors using cells, it is preferable to use cellsexpressing not only PGES but those simultaneously expressing COX. Inaddition, a phenomenon where both enzymes function cooperatively(coupling) was demonstrated in cells expressing both of COX-1 andPGES-1, and in cells expressing both of COX-2 and PGES-2. Accordingly,actually in vivo, during PGE2 production induced by the stimulusaccompanying inflammation, both of COX-2 and PGES-2, or both of COX-1and PGES-1, are indicated to have an important role functioningcooperatively. Thus, cells expressing these combinations seem toreproduce the situation caused by an inflammatory stimulus in astabilized form. Furthermore, cell proliferation in addition to PGE2production is increased by the coexpression of PGES-2 and COX-2 (FIG.12), which may reflect an aspect of a process where normal cellstransform into cancer cells.

INDUSTRIAL APPLICABILITY

The present invention provides a PGES-1 protein having PGE2 synthaseactivity and genes encoding the protein, which enables the efficientproduction of PGE2 as well as the efficient screening of PGE2 synthaseinhibitors that are useful as anti-inflammatory drugs, and such.Specifically, efficient and accurate screening of PGE2 synthaseinhibitors is enabled by cells simultaneously expressing the PGE2synthase and COX.

1-19. (canceled)
 20. A host cell harboring a vector comprising anucleotide sequence encoding a COX enzyme and a vector comprising anucleotide sequence encoding a polypeptide selected from the groupconsisting of: (a) a polypeptide comprising SEQ ID NO: 1; (b) apolypeptide comprising SEQ ID NO: 1 with between one and thirty aminoacid substitutions, deletions, additions, or insertions, wherein thepolypeptide has PGE2 synthase activity; (c) a polypeptide encoded by aDNA that hybridizes under stringent conditions to a DNA consisting ofthe complement of SEQ ID NO: 2; and (d) PGES-2.
 21. The host cell ofclaim 20, wherein the polypeptide comprises SEQ ID NO: 1 and the COXenzyme is COX-1.
 22. The host cell of claim 20, wherein the polypeptidecomprises SEQ ID NO: 1 with between one and thirty amino acidsubstitutions, deletions, additions, or insertions, wherein thepolypeptide has PGE2 synthase activity, and the COX enzyme is COX-1. 23.The host cell of claim 20, wherein the polypeptide is encoded by a DNAthat hybridizes under stringent conditions to a DNA consisting of thecomplement of SEQ ID NO: 2, and the COX enzyme is COX-1.
 24. The hostcell of claim 20, wherein the polypeptide is PGES-2 and the COX enzymeis COX-2.
 25. A method of screening for a PGH2 synthase inhibitor, themethod comprising the steps of: (1) contacting a test compound and PGH2with a host cell harboring a vector comprising a nucleotide sequenceencoding a polypeptide selected from the group consisting of: (a) apolypeptide comprising SEQ ID NO: 1; (b) a polypeptide comprising SEQ IDNO: 1 with between one and thirty amino acid substitutions, deletions,additions, or insertions, wherein the polypeptide has PGE2 synthaseactivity; and (c) a polypeptide encoded by a DNA that hybridizes understringent conditions to a DNA consisting of the complement of SEQ ID NO:2; (2) detecting PGE2 produced by the host cell; and (3) selecting atest compound that reduces the level of PGE2 produced as compared withthat produced in the absence of the test compound.
 26. The method ofclaim 25, wherein the polypeptide comprises SEQ ID NO:
 1. 27. The methodof claim 25, wherein the polypeptide comprises SEQ ID NO: 1 with betweenone and thirty amino acid substitutions, deletions, additions, orinsertions, wherein the polypeptide has PGE2 synthase activity.
 28. Themethod of claim 25, wherein the polypeptide is encoded by a DNA thathybridizes under stringent conditions to a DNA consisting of thecomplement of SEQ ID NO:
 2. 29. A method of screening for a PGE2synthase inhibitor, comprising the steps of: (1) contacting the hostcell of claim 21 with a test compound and arachidonic acid; (2)detecting the level of PGE2 produced by the host cell; and (3) selectinga test compound that reduces the level of PGE2 produced as compared withthat produced in the absence of the test compound.
 30. A method ofscreening for a PGE2 synthase inhibitor, comprising the steps of: (1)contacting the host cell of claim 22 with a test compound andarachidonic acid; (2) detecting the level of PGE2 produced by the hostcell; and (3) selecting a test compound that reduces the level of PGE2produced as compared with that produced in the absence of the testcompound.
 31. A method of screening for a PGE2 synthase inhibitor,comprising the steps of: (1) contacting the host cell of claim 23 with atest compound and arachidonic acid; (2) detecting the level of PGE2produced by the host cell; and (3) selecting a test compound thatreduces the level of PGE2 produced as compared with that produced in theabsence of the test compound.
 32. A method of screening for a PGE2synthase inhibitor, comprising the steps of: (1) contacting the hostcell of claim 24 with a test compound and arachidonic acid: (2)detecting the level of PGE2 produced by said host cell; and (3)selecting a test compound that reduces the level of PGE2 produced ascompared with that produced in the absence of the test compound.
 33. Akit for screening for a PGE2 synthase inhibitor, the kit comprising:PGH2 and a host cell harboring a vector comprising a nucleotide sequenceencoding a polypeptide selected from the group consisting of: (a) apolypeptide comprising SEQ ID NO: 1; (b) a polypeptide comprising SEQ IDNO: 1 with between one and thirty amino acid substitutions, deletions,additions, or insertions, wherein the polypeptide has PGE2 synthaseactivity; and (c) a polypeptide encoded by a DNA that hybridizes understringent conditions to a DNA consisting of the complement of SEQ ID NO:2.
 34. The kit of claim 33, wherein the polypeptide comprises SEQ IDNO:
 1. 35. The kit of claim 33, wherein the polypeptide comprises SEQ IDNO: 1 with between one and thirty amino acid substitutions, deletions,additions, or insertions, wherein the polypeptide has PGE2 synthaseactivity.
 36. The kit of claim 33, wherein the polypeptide is encoded bya DNA that hybridizes under stringent conditions to a DNA consisting ofthe complement of SEQ ID NO:
 2. 37. A kit for screening for a PGE2synthase inhibitor, the kit comprising the host cell of claim 21 andarachidonic acid.
 38. A kit for screening for a PGE2 synthase inhibitor,the kit comprising the host cell of claim 22 and arachidonic acid.
 39. Akit for screening for a PGE2 synthase inhibitor, the kit comprising thehost cell of claim 23 and arachidonic acid.
 40. A kit for screening fora PGE2 synthase inhibitor, the kit comprising the host cell of claim 24and arachidonic acid.